Cell death is an integral part of the immune responses to pathogenic infection, and different forms of cell death can have different immunological consequences. For example, apoptosis is thought to clear viral infected cells and to control run-away immune responses whereas pyroptosis of macrophages can result in the release of cytokines. The Tumor Necrosis Factor Receptor (TNF-R) family of death receptors can trigger apoptosis but also an alternative form of death called programmed necrosis or necroptosis. Necroptosis is dependent on the RIP1 and RIP3 kinases but little is known on its physiological role. To address this, we have generated three lines of tissue- specific FADD knockout mice. FADD is a known adapter protein for all the TNF-R death receptors and thus FADD-deficient cells are resistant to death receptor-mediated apoptosis. However, stimulation of FADD-deficient cells leads to death through necroptosis. This occurs in T-cell receptor stimulated T cells from T-cell specific-FADD knockout (tFADD-/-) mice, in lipopolysaccharide (LPS) stimulated dendritic cells (DCs) from DC-specific-FADD deficient (dcFADD-/-) mice and in LPS stimulated macrophages from macrophage-specific FADD deficient (mFADD-/-) mice. T cells from tFADD-/- mice are functionally defective due to premature necroptosis. However, FADD-deficient DCs are functionally normal and can secrete cytokines when stimulated. Analysis of dcFADD-/- mice showed that some of their phenotypes are surprisingly similar to DC-less mice with imbalance erythrocytes and myeloproliferative disease. At the same time, dcFADD-/- mice exhibit a modest increase of inflammation. In contrast to other DC-specific apoptosis-resistant mice, aged dcFADD-/- mice don't suffer from autoimmunity but they appear to have an enhanced immune system. We hypothesize that necroptosis is one of the strategies for innate immune cells to stimulate the immune system in cases when receptor-induced apoptosis is blocked. In dcFADD-/- mice, necroptotic DCs releasing inflammatory contents can lead to improved immunity against pathogenic infection. This hypothesis will be tested in aim 1. Using DC-specific FADD-/-/MyD88-/- and dcFADD-/-/RIP3-/- mice, we will address whether constitutive stimulation of DCs through TLRs leads to necroptosis and release of inflammatory contents. To examine the possible enhanced immunity of dcFADD-/- mice, they will be challenged with influenza and Toxoplasma gondii and studied in details. Whether necroptosis is required for immunity against these two pathogens will be assessed using RIP3-/- mice. In Aim 2, we will unravel the biochemical signaling pathways involving FADD in necroptosis in response to TLR stimulation. Successful completion of these aims will greatly enhance our understanding of the host- pathogen interaction and the physiological role of necroptosis. ! ! ! PUBLIC HEALTH RELEVANCE: Cell death, including apoptosis, pyroptosis, autophagic cell death and necrosis, is an integral part of development of the mouse/human immune system and/or responses to pathogenic infection. Members of the tumor necrosis factor (TNF) death receptors can initiate apoptosis and a new form of necrosis called necroptosis but the physiological role of necroptosis is poorly understood; we propose in this application to understand the role of necroptosis in infection by studying mouse models deficient in an adapter protein required for the TNF cell death signals.